Sergey Khorobrykh

Participation of photosynthetic electron transport in production and scavenging of reactive oxygen species.

The photosynthetic electron transport chain (PETC) is the principal place of appearance of reactive oxygen species (ROS) in plants under illumination. The peculiarities of this process in different segments of the PETC are discussed. Oxygen uptake observed under impaired electron donation to photosystem II is attributed mainly to hydroperoxide formation by reaction of oxygen with organic radicals generated after detachment of electrons by P680(+). Oxygen reduction in the plastoquinone pool is suggested to start with the reaction of O(2) with plastosemiquinone, and to be followed by reduction of superoxide to hydrogen peroxide by plastohydroquinone. The distribution of plastoquinone throughout the thylakoid membrane interior provides for the generation of ROS by this route all along the membrane surface. O(2) reduction at the acceptor side of photosystem I remains poorly understood. The regeneration of antioxidants is stated to be a priority task of photosynthetic electron transport in view of the effectiveness of monodehydroascorbate as electron acceptor. We propose that ROS generation in the plastoquinone pool and the possible formation of hydroperoxides in the vicinity of photosystem II are key processes participating in the primary stages of redox signaling.

Kinetics of the plastoquinone pool oxidation following illumination Oxygen incorporation into photosynthetic electron transport chain.

The oxidation of the PQ‐pool after illumination with 50 or 500 μmol quanta m−2 s−1 was measured in isolated thylakoids as the increase in ΔA 263, i.e., as the appearance of PQ. While it was not observed under anaerobic conditions, under aerobic conditions it was biphasic. The first faster phase constituted 26% or 44% of total reappearance of PQ, after weak or strong light respectively. The dependence on oxygen presence as well as the correlation with the rate of oxygen consumption led to conclusion that this phase represents the appearance of PQ from PQ ▪− produced in the course of PQH2 oxidation by superoxide accumulated in the light within the membrane.

Photoproduction of catalase-insensitive peroxides on the donor side of manganese-depleted photosystem II: evidence with a specific fluorescent probe.

The photoproduction of organic peroxides (ROOH) in photosystem II (PSII) membranes was studied using the fluorescent probe Spy-HP. Two types of peroxide, highly lipophilic ones and relatively hydrophilic ones, were distinguished by the rate of reaction with Spy-HP; the former oxidized Spy-HP to the higher fluorescent form Spy-HPOx within 5 min, while the latter did so very slowly (the reaction was still not completed after 180 min). The level of photoproduction of these peroxides was significantly larger in the alkaline-treated, Mn-depleted PSII membranes than that in the untreated membranes, and it was suppressed by an artificial electron donor (diphenylcarbazide or ferrocyanide) and by the electron transport inhibitor diuron. Postillumination addition of Fe(2+) ions, which degrade peroxides by the Fenton mechanism, abolished the accumulation of Spy-HPOx, but catalase did not change the peroxide level, indicating that the detected species were organic peroxides, excluding H(2)O(2). These results agreed with our previous observation of an electron transport-dependent O(2) consumption on the PSII donor side and indicated that ROOH accumulated via a radical chain reaction that started with the formation of organic radicals on the donor side. Illumination (λ > 600 nm; 1500 μmol of photons m(-2) s(-1)) of the Mn-depleted PSII membranes for 3 min resulted in the formation of nearly 200 molecules of hydrophilic ROOH per reaction center, but only four molecules of highly lipophilic ROOH. The limited formation of the latter was due to the limited supply of its precursor to the reaction, suggesting that it represented structurally fixed peroxides, i.e., either protein peroxides or peroxides of the lipids tightly bound to the core complex. These ROOH forms, likely including several species derived from lipid peroxides, may mediate the donor side-induced photoinhibition of PSII via protein modification.

Photoconsumption of oxygen in photosystem II preparations under impairment of the water-oxidizing complex.

Oxygen consumption in photosystem II (PSII) preparations in the light was 2 μmol O2/h per mg Chl at weakly acidic and at neutral pH values. It increased fourfold to fivefold at pH 8.5-9.0. The addition of either artificial electron donors for PSII such as MnCl2 or diphenylcarbazide, or diuron as an inhibitor of electron transfer from QA, the primary bound quinone acceptor, to QB, the secondary bound quinone acceptor of PSII, resulted in a decrease in oxygen consumption rate at basic pH to value close to ones measured at pH 6.5. Such additions did not affect oxygen consumption at lower pH values. The induction of variable chlorophyll fluorescence yield in the light differed greatly at pH 6.5 and 8.5. While at pH 6.5 the fluorescence yield, after an initial fast rise almost to Fmax, only slightly decreased, at pH 8.5 after such a rise it dropped promptly to a low value. The additions of the artificial electron donors at pH 8.5 resulted in the induction kinetics close to that observed at pH 6.5. These data indicate impairment of electron donation to P680+ that could be caused by damage to the water oxidation system at basic pH values. In experiments with PSII preparations treated with Tris to destroy the water-oxidizing complex, photoconsumption of oxygen in the entire pH region was close to the values in untreated preparations at basic pH. In untreated preparations the rate of light-induced oxygen consumption decreased in the presence of catalase, which decomposes H2O2, as well as in the presence of electron acceptor potassium ferricyanide. From these data it is suggested that the light-induced oxygen consumption in PSII is caused by two processes, by an interaction of O2 with organic radicals, which were formed due to oxidation of components of the donor side of this photosystem (proteins, lipids, pigments) by cation-radical P680+, as well as by oxygen reduction by still unidentified components of PSII.

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments.

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O(2) photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O(2) photoreduction on the acceptor side of PSII, there is light-induced O(2) consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O(2) uptake on the donor side of PSII is related to interaction of O(2) with radicals produced by photooxidation of organic molecules. The study of flash-induced O(2) uptake finds that removal of Mn from the WOC leads to O(2) photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O(2) evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O(2) uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10microM, corresponding to 2-4 Mn per RC) of Mn(2+), while at higher concentrations (>100microM) Mn(2+) inhibits the O(2) photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn(2+)) leads to both suppression of flash-induced O(2) uptake and disappearance of the Mn-induced activation of the O(2) photoconsumption. We assume that the light-induced O(2) uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O(2) or its reactive forms in the formation of the inorganic core of the WOC.

Reactive oxygen species: Reactions and detection from photosynthetic tissues.

Reactive oxygen species (ROS) have long been recognized as compounds with dual roles. They cause cellular damage by reacting with biomolecules but they also function as agents of cellular signaling. Several different oxygen-containing compounds are classified as ROS because they react, at least with certain partners, more rapidly than ground-state molecular oxygen or because they are known to have biological effects. The present review describes the typical reactions of the most important ROS. The reactions are the basis for both the detection methods and for prediction of reactions between ROS and biomolecules. Chemical and physical methods used for detection, visualization and quantification of ROS from plants, algae and cyanobacteria will be reviewed. The main focus will be on photosynthetic tissues, and limitations of the methods will be discussed.

Volatile Oxylipins and Related Compounds Formed Under Stress in Plants

Plants form volatile oxylipins and related compounds under stress. Some of them are important flavor chemicals and give big impact on the flavor quality of food made from plant materials. They are also involved in defense responses of plants against pathogens and herbivores. Furthermore, in some instances, they cause harmful effects on plants themselves. Because of these significances of volatile oxylipins and related compounds, demands to perform comprehensive analyses of these compounds are increasing. In this chapter, we describe the simple but efficient procedures to reveal profiles of volatile oxylipins and related compounds by using HPLC and GC-MS. They are simple and can be performed in biochemical laboratories equipped with common facilities.

Experimental evidence suggesting that H2O2 is produced within the thylakoid membrane in a reaction between plastoquinol and singlet oxygen

Plastoquinol (PQH2‐9) and plastoquinone (PQ‐9) mediate photosynthetic electron transfer. We isolated PQH2‐9 from thylakoid membranes, purified it with HPLC, subjected the purified PQH2‐9 to singlet oxygen (1O2) and analyzed the products. The main reaction of 1O2 with PQH2‐9 in methanol was found to result in formation of PQ‐9 and H2O2, and the amount of H2O2 produced was essentially the same as the amount of oxidized PQH2‐9. Formation of H2O2 in the reaction between 1O2 and PQH2‐9 may be an important source of H2O2 within the lipophilic thylakoid membrane.

Plastoquinol generates and scavenges reactive oxygen species in organic solvent: Potential relevance for thylakoids

The present work reports reactions of plastoquinol (PQH2-9) and plastoquinone (PQ-9) in organic solvents and summarizes the literature to understand similar reactions in thylakoids. In thylakoids, PQH2-9 is oxidized by the cytochrome b6/f complex (Cyt b6/f) but some PQH2-9 is also oxidized by reactions in which oxygen acts as an electron acceptor and is converted to reactive oxygen species (ROS). Furthermore, PQH2-9 reacts with ROS. Light enhances oxygen-dependent oxidation of PQH2-9. We examined the oxidation of PQH2-9 via dismutation of PQH2-9 and PQ-9 and scavenging of the superoxide anion radical (O2-) and hydrogen peroxide (H2O2) by PQH2-9. Oxidation of PQH2-9 via dismutation to semiquinone was slow and independent of pH in organic solvents and in solvent/buffer systems, suggesting that intramembraneous oxidation of PQH2-9 in darkness mainly proceeds via reactions catalyzed by the plastid terminal oxidase and cytochrome b559. In the light, oxidation of PQH2-9 by singlet oxygen and by O2- formed in PSI contribute significantly. In addition, Cyt b6/f forms H2O2 with a PQH2-9 dependent mechanism. Measurements of the reaction of O2- with PQH2-9 and PQ-9 in acetonitrile showed that O2- oxidizes PQH2-9, forming PQ-9 and several PQ-9-derived products. The rate constant of the reaction between PQH2-9 and O2- was found to be 104 M-1 s-1. H2O2 was found to oxidize PQH2-9 to PQ-9, but failed to oxidize all PQH2-9, suggesting that the oxidation of PQH2-9 by H2O2 proceeds via deprotonation mechanisms producing PQH--9, PQ2--9 and the protonated hydrogen peroxide cation, H3O2+.

Involvement of the chloroplast plastoquinone pool in the Mehler reaction

Light-dependent oxygen reduction in the photosynthetic electron transfer chain, i.e. the Mehler reaction, has been studied using isolated pea thylakoids. The role of the plastoquinone pool in the Mehler reaction was investigated in the presence of dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-INT), the inhibitor of plastohydroquinone oxidation by cytochrome b6/f complex. Oxygen reduction rate in the presence of DNP-INT was higher than in the absence of the inhibitor in low light at pH 6.5 and 7.6, showing that the capacity of the plastoquinone pool to reduce molecular oxygen in this case exceeded that of the entire electron transfer chain. In the presence of DNP-INT, appearance of superoxide anion radicals outside thylakoid membrane represented approximately 60% of the total superoxide anion radicals produced. The remaining 40% of the produced superoxide anion radicals was suggested to be trapped by plastohydroquinone molecules within thylakoid membrane, leading to the formation of hydrogen peroxide (H2 O2 ). To validate the reaction of superoxide anion radical with plastohydroquinone, xanthine/xanthine oxidase system was integrated with thylakoid membrane in order to generate superoxide anion radical in close vicinity of plastohydroquinone. Addition of xanthine/xanthine oxidase to the thylakoid suspension resulted in a decrease in the reduction level of the plastoquinone pool in the light. The obtained data provide additional clarification of the aspects that the plastoquinone pool is involved in both reduction of oxygen to superoxide anion radicals and reduction of superoxide anion radicals to H2 O2 . Significance of the plastoquinone pool involvement in the Mehler reaction for the acclimation of plants to light conditions is discussed.